The barley chromosome in wheat was identified by genomic in situ hybridization (GISH) in which biotin labelled total genomic DNA of barley Betzes was used as probe and the unlabelled total DNA of common wheat Chinese Spring (CS) as blocking DNA. A series of wheat materials were tested as follows: two disomic alien substitution and monosomic alien addition lines, 2n = 43; two monosomic alien substitution lines, 2n = 42; seven disomic alien substitution lines, 2n = 42. RFLP probe psr131 on the short arm of the homologous group 2 was used to analyze the barley chromosome in wheat. The result indicated that there was a same band in barley Betzes and substitution line A5. The chromosome 2A of A5 was substituted by the chromosome 2H of barley. These materials will be useful in transferring the valuable genes in the chromosome 2H to wheat.